P397: PROLACTIN RECEPTOR CONFERS CHEMORESISTANCE DUE TO SENESCENCE ENTRANCE IN ACUTE MYELOID LEUKEMIA
Authors: L. Cuesta-Casanovas, J. M. Cornet-Masana, J. M. Carbó, J. Delgado-Martínez, L. Clément-Demange, A. Banús-Mulet, F. Guijarro, J. Esteve, R. M. Risueño
Journal: HemaSphere (2022)

Background: Acute myeloid leukaemia (AML) is a hematopoietic neoplasm characterized by frequent relapse because of chemotherapy resistance, which confers poor outcome. For this reason, the study of new therapeutic approaches that completely eradicate the disease is an urgent need. A previous in silico analysis performed in our laboratory demonstrated that PRLR signalling pathway is overactivated in leukemic stem cells (LSCs) in comparison to hematopoietic stem cells (HSCs), suggesting a relationship between PRLR and AML resistance and progression. Aims: The main objective was to decipher the role of PRLR and its signalling pathway in the transformation events associated with the initiation and maintenance of AML. Methods: mRNA expression of PRLR and Stat5a was analysed from public repositories. Surface PRLR expression was assessed by flow cytometry and total protein expression by western blot. Stat5 activation (phosphorylation status) and induced gene regulation were determined in AML cells ectopically expressing the wildtype PRLR (PRLRwt) and the dominant negative isoform (PRLRsh) and treated with its natural ligand (PRL) or an antagonist (G129R-PRL), by western blot and luciferase reporters. Senescence status was analysed by SA-β-galactosidase staining by microscopy and quantified using ImageJ software. Cytarabine resistant cells (AraC-R) were obtained by treating them during several weeks with increasing doses of cytarabine. PRLR expression was silenced using the CRISPR-Cas9 technology. Clonogenicity was studied in AML samples and healthy linage-depleted cord blood cells treated with PRL and hPRL-G129R and seeded in a semisolid medium in presence of instructive cytokines. Results: AML patient cells express higher levels of PRLR in terms of mRNA, protein surface expression and total protein in comparison to healthy donors. Stat5 mRNA was overexpressed in AML, and PRL induced its phosphorylation and activation in presence of PRLR wt isoform, while the dominant negative did not respond to PRL. The antagonist of the receptor did not induce any effect. Stat5 mRNA expression was increased at relapse, and so it was the PRLR surface and total protein when AML cells acquire chemoresistance. Moreover, PRLR wildtype cells showed greater levels of SA-β-galactosidase, an indicative of the entrance in senescence. This effect was reverted when PRLR was knocked-down using CRISPR-Cas9 technology. The inhibition of PRLR with an antagonist or its downregulation with the CRISPR-Cas9 technology decreased clonogenicity capacity of AML patient cells, without affecting healthy donor cells. Summary/Conclusion: PRLR expression is higher in AML patient samples, and correlates with chemoresistance and refractoriness, making the receptor an adequate biomarker for AML diagnosis and follow-up. PRLR-PRL signalling promotes the activation of Stat5, which is increased in AML samples, especially at relapse, suggesting an important role in AML progression and chemoresistance, a process related with increasing levels of PRLR surface expression. Moreover, the suppression of PRLR expression sensitizes the cells to chemotherapy. Those results demonstrate for the first time that PRLR suppression increases the sensitivity to chemotherapy and decreases the clonogenicity capacity of AML cells, suggesting the use of antagonists of the receptor as new therapeutic strategies against AML.